Respiratory Syncytial Virus Two-Step Infection Screen Reveals Inhibitors of Early and Late Life Cycle Stages

ABSTRACT Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available. We aimed to discover new and chemically diverse candidates to enrich the hRSV drug development pipeline. We used a two-step screen that interrogates compound efficacy after primary infection and a consecutive virus passaging. We resynthesized selected hit molecules and profiled their activities with hRSV lentiviral pseudotype cell entry, replicon, and time-of-addition assays. The breadth of antiviral activity was tested against recent RSV clinical strains and human coronavirus (hCoV-229E), and in pseudotype-based entry assays with non-RSV viruses. Screening 6,048 molecules, we identified 23 primary candidates, of which 13 preferentially scored in the first and 10 in the second rounds of infection, respectively. Two of these molecules inhibited hRSV cell entry and selected for F protein resistance within the fusion peptide. One molecule inhibited transcription/replication in hRSV replicon assays, did not select for phenotypic hRSV resistance and was active against non-hRSV viruses, including hCoV-229E. One compound, identified in the second round of infection, did not measurably inhibit hRSV cell entry or replication/transcription. It selected for two coding mutations in the G protein and was highly active in differentiated BCi-NS1.1 lung cells. In conclusion, we identified four new hRSV inhibitor candidates with different modes of action. Our findings build an interesting platform for medicinal chemistry-guided derivatization approaches followed by deeper phenotypical characterization in vitro and in vivo with the aim of developing highly potent hRSV drugs.

n.c., not calculable by the used curve fit algorithm; n.a., not applicable; 1 dose based on IC90 round II; 2 114 SA- Table 3

General information
This document describes the general procedures, experimental details as well as characterizations of chemically synthesized compounds 3s, 5s, (R)-9s, (S)-9s, and 12s. Furthermore, details regarding the performed purity analyses of screening compounds are included.
Microwave-assisted (mw) syntheses were carried out in a Discover microwave synthesis system from CEM. For column chromatography, either the automated flash column chromatography (AFC) system CombiFlash Rf 150 (Teledyne Isco) equipped with RediSepRf silica columns was used or manual flash column chromatography (MFC) with Silica 60 M, 0.04 -0.063 mm or 0.063 -0.2 mm, (Macherey-Nagel) was performed.
Final products were dried under high vacuum. In case the final compound was purified with semipreparative high performance liquid chromatography (semi-prep HPLC), the corresponding isolated fraction was lyophilized using a Christ Alpha 2-4 LD plus freeze-dryer connected to Chemistry Hybrid Pump RC6 (Vacuubrand). For semi-prep HPLC, an Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific) equipped with Dionex RS Pump, Diode Array Detector, Automated Fraction Collector, Nucleodur C18 Gravity column (250 mm x 10 mm (column A) or 16 mm (column B), particle size 5 µm) was used.
High resolution mass spectrometry (HRMS) measurements were conducted with a Q Exactive Focus (Thermo Fischer Scientific) connected to Dionex Ultimate 3000 RS Pump and Autosampler as well as UHPLC system Column compartment with Nucleodur C18 Pyramid column (150 mm x 2 mm, particle size 3 µm) and Diode Array Detector.

General procedure A (GP A)
The general procedure A was performed as described in the protocol of Cheng et al. 1 . The corresponding amine (1.0 -2.2 equivalents (equiv.)) and N,N-diisopropylethylamine (DIPEA, 2.2 -3.0 equiv.) were added dropwise to a solution of the related starting material in dry dichloromethane (DCM, 0.54 M) at 0 °C. The mixture was stirred at the same temperature or reaching room temperature. After a full conversion detected via LC-MS, the reaction mixture was washed with hydrochloric acid (HCl, 2 M, aqueous (aq.)). From this aqueous layer, the crude was extracted twice with DCM. After combining all organic layers and drying them over MgSO4, the solvent was removed in vacuo. The crude was purified via AFC to isolate the related product.

General Procedure C (GP C)
In case of a Boc-protected starting material, the compound was first dissolved in DCM (0.1 M), cooled to 0 °C and treated with HCl (4 N in 1,4-dioxane, 10.0 equiv.). The mixture was stirred overnight reaching room temperature. After removing the solvent, the resulting HCl salt was directly used for the substitution reaction. 9b-1 or deprotected 9b-2, respectively, triethylamine (NEt3, 1.1 equiv. for amines or 2.2 equiv. for HCl salts), and 9d (1.0 equiv.) were stirred in tetrahydrofuran (THF, 0.45 M) at room temperature for 1 -2 days. Then, the reaction mixture was poured into ice and the formed beige solid was extracted with DCM. After drying the organic layer over MgSO4, the solvent was removed in vacuo.
The obtained crude product was purified via AFC and/or semi-prep HPLC 3 .
The reaction mixture was cooled to room temperature and poured into ice water. The formed, white to yellow solid was filtered, washed with water, transferred with acetone and dried in vacuo to obtain 3b